The long-term objectives of the application are to understand the structure, mechanism and biosynthesis of the human multidrug resistance P-glycoprotein (P-gp). This knowledge will then be used to develop strategies to shut off the transporter during cancer chemotherapy or treatment of AIDS. P-gp interferes in the treatment of cancer and AIDS by preventing anticancer drugs or HIV-1 protease inhibitors from reaching their targets. Efforts to develop effective inhibitory drugs that specifically bind to P-gp have been hampered by the lack of structural information about the drug-binding domain and the conformational changes that take place during transport. These issues will be addressed by testing the hypothesis that transmembrane segments 5, 6, 11 and 12 of P-gp form the drug-binding domain and that they undergo conformational changes during drug transport. These hypotheses will be tested using a Cys-less mutant of P-gp together with cysteine-scanning mutagenesis, thiol-reactive substrates and disulfide crosslinking. A new approach will also be developed to inhibit P-gp function by inhibiting folding and exit of P-gp from the endoplasmic reticulum. Preliminary evidence suggests that some compounds can block the conversion of a biosynthetic intermediate of P-gp into mature enzyme.